Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. Various methods exist for detecting and measuring intracellular cAMP, but none are ideally suited for monitoring spatial and temporal distributions of cAMP in living cells. For example, radioimmunoassay or enzyme immunoassays for measuring cAMP require destroying large amounts of cells or tissue, have very poor spatial and temporal resolution, and measure total rather than free cAMP. Use of engineered cyclic nucleotide-gated channels to detect free cAMP provides good temporal resolution and quantification but uses indirect calcium measurements or nontrivial patch-clamp techniques and lacks the flexibility of measuring cAMP changes within various subcellular compartments (Rich et al., Proc. Natl. Acad. Sci. USA 98, 13049-54, 2001; Rich et al., J. Gen. Physiol. 116, 147-61, 2000). Free cAMP can be imaged in single cells microinjected with fluorophore-labeled C and R subunits (Adams et al., Nature 349, 694-97, 1991) or in cells expressing two colors of GFP mutants fused to the C and R subunits (Zaccolo et al., Nat. Cell Biol. 2, 25-29, 2000), which dissociate from each other and lose fluorescence resonance energy transfer upon elevation of cAMP. However, the expression levels of the two fusions have to be carefully matched to allow reliable measurement. Even so, mixed tetramerization may occur between the fluorophore-attached subunits and endogenous partners, reducing the number of functional reporter molecules. Furthermore, it can be difficult to target such bimolecular reporters to different subcellular locations while maintaining appropriate stoichiometry.
There is a need in the art for sensitive cAMP reporters and methods which can be used for accurate measurements of spatial and temporal cAMP distributions in living cells.